Antibiotic,negamycin,and processes for the preparation thereof

ABSTRACT

PROCESS FOR THE PRODUCTION OF AN ANTIBIOTIC SUBSTANCE CALLED NEGAMYCIN WHICH COMPRISES CULTIVATING A STRAIN OF STREPTOMYCES PURPEOFUSCUS A.T.C.C. 21470 IN AN AQUEOUS CARBOHYDRATE SOLUTION CONTAINING A NITROGENOUS NUTRIENT UNDER AEROBIC CONDITIONS UNTIL SUBSTANTIAL ANTIBACTERIAL ACTIVITY IS IMPARTED TO SAID SOLUTION AND THEN RE-   COVERING SAID NEGAMYCIN FROM SAID SOLUTION AND NEGAMYCIN WHICH IS EFFECTIVE FOR INHIBITING PSUEDOMONAS, SALMONELLA, SHIGELLA, KLEBSIELLA, E. COLI, STAPHYLOCOCCI AND WHICH HAS THE FOLLOWING STRUCTURE: H2N-CH2-CH(-OH)-CH2-CH(-NH2)-CH2-CO-NH-N(-CH3)-CH2-COOH

y 3, 1973 HAMAO UMEZAWA ET AL 3,743,580

NEW ANTIBIOTIC, NEGAMYCIN, AND PROCESSES FOR THE PREPARATION THEREOFOriginal Filed Apnl 28, 1970 2 Sheets-Sheet l IIOO O N F o 0 3 LL. I 8

ID K 0 m m N o o o o o o 0 Q 00 w r cu r July 3, 1973 HAMAO z w ET AL3,743,580

NEW ANTIBIOTIC. NEGAMYCIN, AND PROCESSES FOR THE PREPARATION THEREOFOriginal Filed April 28, 1970 2 Sheets-Shunt 2 0 8 LI. N

H q T 8 .12 m If) United States Patent 3,743,580 ANTIBIOTIC, NEGAMYCIN,AND PROCESSES FOR THE PREPARATION THEREOF Hamao Umezawa, Tokyo, ShinichiKondo, Yokohama, and Keuji Maeda, Tomio Takeuchi, and Masa Hamada,Tokyo, Japan, assignors to Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai,Kami-Osaki, Shinagawa-ku, Tokyo, Japan Original application Apr. 28,1970, Ser. No. 32,641, now Patent No. 3,679,742. Divided and thisapplication May 15, 1972, Ser. No. 253,618

Int. Cl. C12b 1/00 US. Cl. 195-80 R 7 Claims ABSTRACT OF THE DISCLOSUREProcess for the production of an antibiotic substance called negamycinwhich comprises cultivating a strain of Streptomyces purpeofuscusA.T.C.C. 21470 in an aqueous carbohydrate solution containing anitrogenous nutrient under aerobic conditions until substantialantibacterial activity is imparted to said solution and then recoveringsaid negamycin from said solution and negamycin which is eifective forinhibiting Psuedomonas, Salmonella, Shigella, Klebsiella, E. coli,Staphylococci and which has the following structure:

This is a division of application Ser. No. 32,641, filed Apr. 28, 1970,now Pat. No. 3,679,742.

This invention relates to a new useful antibiotic substance callednegamycin, and to its production. More particularly, it relates toprocesses for its production by fermentation and methods for recoveryand purification. This invention embraces this antibacterial agent andits salts in dilute solutions, as crude concentrates, as crude solidsand as pure solid. This substance is effective in inhibition ofPseudomonas, Salmonella, Klebsiella and Staphylococci. This substancehas low toxicity and exhibits therapeutic effect on infections ofPseudomonas and other sensitive organisms in mice. This substance isuseful in the cure of infections of Pseudomonas and other sensitiveorganisms in humans and animals.

There is now provided, according to the present invention, an antibioticsubstance (and its salts) effective in inhibiting Pseudomonas,Salmonella, Klebsiella and Staphylococci, said antibiotic being solublein water and practically insoluble in methanol, ethanol, butanol, ethylacetate, butyl acetate, chloroform, and benzene, exhibiting no maximumabsorption of ultraviolet light from 220-400 mg, giving positivereactions to Ninh'ydrin, red tetrazolium and Rydon-Smith and negativereactions to Sakaguchi and Molisch, exhibiting absorption bands in theinfrared region of the spectrum when pelleted with potassium bromide atthe following wave numbers in cm.- 3430, 3200, 3050, 2950, 1660, 1590,1405, 1320, 1140, 1050, 970, "890, 820, 720, being optically active, [a]=+2.5 (c. 2, H O), having the molecular formula of C H N O showing threepK values of 3.55, 8.10, 9.75, and having the following structure:

Referring to the drawings: FIG. 1 is the infrared absorption spectrum ofnegamycin taken with potassium bromide, and FIG. 2 is the nuclearmagnetic resonance specrum of negamycin in D 0.

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There is provided according to the present invention the process for theproduction of the antibiotic negamycin which comprises cultivating astrain of S. purpeofuscus in a nutrient medium under aerobic conditionsuntil a substantial amount of negamycin is accumulated in said solution.

Negamycin is a new antibiotic discovered by the present inventors. Theorganism producing the antibiotic of the present invention was firstfound by the present inventors and was isolated from a soil samplecollected at Myogisan, Gunma prefecture in August, 1964 and was giventhe laboratory number M890-C2. The inventors also isolated negamycinfrom other two strains which were given the laboratory numbers of No.MA91-Ml and No. MA104-M1. The strain No. MA91-M1 was isolated from asoil sample collected at Nojiriko, Nagano prefecture in January 1965,and the strain No. MA104-Ml was isolated from a soil sample collected atSakomachi, Tokushima city in February 1965. These strains have manycommon properties with the strain No. M890-C2 and also with Streptomycespurpeofuscus. Though there were some minor differences among them, thesestrains were classified as Sfrept'omyces purpeofuscus. Moreover, a typeculture of S. purpeofuscus (S. pwrpeofuscus Yamaguchi and Saburi ISP5283, ISP being the abbreviation of International Streptomyces Project)was confirmed by the present inventors to produce negamycin. The strainNo. M890-C2 was deposited Kogyogijutsuin Hakko Kenkyujo on May 6', 1969,and the deposit number 306 was given. It was also deposited in AmericanType Culture Collection as ATCC 21470.

The strain No. M89=0-C2 has the following characteristics. Undermicroscopic, well-branched substrate mycelium develops straight toflexuous aerial mycelium without whorl branching nor spiral formation.Surface of spore is smooth.

Characters on various media (Designation of color is described in inaccordance with Color Harmony Manual of Container Corporation ofAmerica):

(1) On glycerol Czapeks agar medium incubated at 27 C.: Pale yellow todark yellowish brown [Spice brown 4 ni] growth forms abundant aerialmycelium of light grey color; brownish to pale reddish brown solublepigment is produced;

(2) On Krainskys glucose asparagine agar medium incubated at 27 C.:Growth of yellowish brown [Luggage Tan 4 ne] to dark yellowish brown[Clove Brown 3 mi] produces aerial mycelium of yellowish white to lightbrownish gray; soluble pigment is faint brown;

(3) On calcium malate agar medium incubated at 27 C.: Growth of grayish'brown [Fawn 5 ig] produces scant grayish white to light gray aerialmycelium. No soluble pigment. Calcium malate around growth issolubilized strongly after 3 days incubation;

(4) In peptone solution containing 1.0% sodium nitrate incubated at 27C.: Colorless to pale yellowish brown growth without aerial mycelium. Nosoluble pigment or brown pigment slightly. Weak reduction of nitrate isob served;

(5) 0n potato plug incubated at 27 C.: Wrinkled growth of pale yellowishbrown [Lt Gold 2 ic] to yellowish brown [Yellow Maple 3 ng], lately darkyellowish brown [Clove Brown 3 pl] to black. No aerial mycelium. Faintbrownish soluble pigment;

(6) On starch agar plate incubated at 27 C.: Pale yellowish brown toyellowish brown [Spice Brown 4 mi] growth produces aerial mycelium ofyellowish white to light gray color. Brownish to pale reddish brownsoluble pigment. Positive hydrolysis of starch;

(7) 0n nutrient agar medium incubated at 37 C.: No growth within 14 daysincubation;

(8) On nutrient agar medium incubated at 27 C.: Pale reddish brown{Brick Red 6 /2 ng] growth produces no aerial mycelium and produces palereddish brown soluble pigment;

(9) On Loefllers coagulated serum medium incubated at 37 C.: No growthwithin 14 days incubation;

(10) On gelatin stub cultured at 20 C.: Colorless to pale yellowishbrown growth produces no aerial mycelium and produces soluble pigment ofbrown to dark brown color. Liquefaction of gelatin is strong;

(11) On skimmed milk incubated at 37 C.: Colorless growth produces noaerial mycelium nor soluble pigment. After complete coagulation of milkat 4 days incubation, slow peptonization occurs;

(12) On tyrosine agar medium incubation at 27 C.: Growth of colorless orgray to dark brown color produces thin aerial mycelium of white tograyish white color. Determination of tyrosinase activity is difficultbecause of blackish pigmentation of soluble pigment;

(13) On cellulose (filter paper) incubated at 27 C.: Scant growthwithout decomposition of cellulose;

(14) Utilization of carbohydrates on Pridham- Gottliebs basal mediumincubated at 27 C.: Positive growth with starch, dextrin, glycerol,galactose, glucose, sucrose, maltose and mannose. Negative growth withinositol, lactose, mannitol, raffinose, rhamnose, inulin, sorbitol,fructose, xylose, arabinose, salicin and dulcitol.

The characters of the strain No. M890-C2 may be summarized as follows:It belongs to genus Streptomyces having aerial mycelium forming neitherspirals nor whorls; the surface of spores is smooth; on various media itforms dark yellowish brown growth with gray or light gray to lightbrownish gray aerial mycelium; it forms no soluble pigment or faintlyreddish brown soluble pigment very slightly; the reverse of the growthturns to black during days of the incubation; on nutrient agar at 27 C.it forms reddish brown growth and reddish brown soluble pigment; itforms melanin pigment, hydrolyzes starch and has relatively strongproteolytic activity. These properties conform to those of Streptomycespurpeofuscus Yamaguchi and Saburi described in International Journal ofSystematic Microbiology, 18, 364, 1968. However, the strain No. M890-C2does not utilize lactose and xylose which are utilized by S.purpeofuscus. On the other hand, another strain No. MAl04-Ml utilizesxylose. Though the strains producing negamycin isolated by the presentinventors had minor differences with known species of S. purpeofuscus,they were assigned to S. purpeofuscus. Moreover, the type culture of S.purpeofuscus (ISP 5283) was confirmed by the inventors to producenegamycin. On production of antibiotics by S. purpeofuscus Yamaguchi andSaburi there has been only simple description (I. of General and AppliedMicrobiology, 1, 201-234, 1955) that its culture filtrate showedinhibition against trichomonas, Gram positive bacteria and acid-fastbacteria. There was no description of production of antibiotics activeagainst Gram negative bacteria. Therefore, though S. purpcofuscus is aknown species, there has never been observation on production ofnegamycin until the present invention.

As it is well known, Streptomyces is generally not stable and easilygives mutants and variants artificially or in nature. Streptomycespurpeofuscus in the present invention includes all of these mutants andvariants which produce negamycin, that is, those which can not beabsolutely differentiated from S. purpeofuscus and produce negamycin.

Method of determining amount of negamycin: It is determined by anordinary cylinder plate method using pure negamycin as the standard andthe sensitive organism such as E. coli K12, Pseudomonas aeruginaosa,etc.

S. purpeofuscus producing negamycin when grown under suitable conditionsproduces negamycin. A fermentation broth containing negamycin isprepared by inoculating spores or mycelium of the negamycin-producingorganism into a suitable medium and then cultivating under aerobiccondition. For the production of negamycin cultivation on a solid mediumis possible, but for production of large quantities cultivation in aliquid medium is preferred. Any fermentation temperature can be employedwithin the range in which the negamycin-producing organism can grow andproduce negamycin, although 25-32" C. is preferred. Media consisting ofknown kinds of nutritional sources for actinomycetes are useful forproduction of negamycin. For example, commercial products such aspepstone, meat extract, N-Z amine, casein, soybean meal, corn steepliquor, peanuts meal, cotton seed meal, sodium nitrate, ammoniumnitrate, ammonium sulfate and other nitrogenous materials such as wheatbran, rice bran, etc. are useful as the nitrogen source. Thecommerically available products, such as glucose, glycerol, starch,maltose, dextrin, sucrose, lactose, soybean oils and other carbohydratesor other fats in pure or crude state are useful as the carbon source.Sodium chloride, sodium or potassium phosphate, calcium carbonate can bealso added. Trace of metal salts can be added, if necessary. Any kindsof constituents which can be utilized by negamycin-producing organismsfor production of negamycin is useful.

The fermentation is continued until negamycin is substantiallyaccumulated. For example, spores and mycelia on the slant culture of S.purpeofuscus were inoculated into a medium consisting of glucose 2.0%,starch 2.0%, soybean meal 2.0%, yeast extract 0.5%, sodium chloride0.25%, calcium carbonate 0.35%, CuSO -5H O 0.0005%, MnCl -7HO 0.0005%,ZnSO -7H O 0.005%, (adjusted to pH 7.0) and shake-cultured at 27 C.Then, the accumulation of negamycin was observed in 3-8 days, forinstance, 20 g/ml. on 3rd day, 108 g/ml. (pH 7.4) on 6th day, 185 g/ml.(pH 7.6) on 7th day, 122 ,lLg-/l11l. (pH 7.8) on 8th day.

Negamycin is relatively stable in its aqueous solution. For instance,after heating of 5 mg./ml. negamycin solution of various pH (that is1.8, 7.2, 9.2) at 60 C. for 30 minutes, 91% remained withoutdecomposition. When 2 mg./ml. of negamycin solution of various pH waskept at 27 C. for a week, the following percent of negamycin remainedwithout decomposition: 86% at pH 2.25, at pH 4.90, 76% at pH 7.15, 89%at pH 9.0, 79% at pH 10.2.

There is provided according to the present invention processes forextraction and purification of negamycin. Negamycin is soluble in waterand exists mainly in the liquid part of the fermentation broth.Negamycin in the broth is not transferred from the broth to organicsolvents such as butanol, ethyl acetate, ether, chloroform, benzene.

With the adsorbents, negamycin can be obtained from the fermented brothor from its aqueous solution. Active carbon is one of preferredadsorbents. Negamycin adsorbed on active carbon can be elutedefficiently by aqueous methanol, aqueous butanol, aqueous acetone. It ismore easily eluted, if the elution is made at acid condition withhydrochloric acid. Though negamycin has carboxylic acid group, havingtwo basic groups the whole molecule is basic and adsorbed by a cationexchange resin. A cation exchange resin process is the most suitablemethod for extraction of negamycin from the fermented broth or itsaqueous solution. All kinds of resins having carboxylic acid group orsulfonic acid group are useful and they can be used in H form, sodiumform potassium form or ammonium form or the mixed form. Negamycinadsorbed on the cation exchange resin can be eluted with an acid such ashydrochloric acid, sulfuric acid, etc., but more preferably with aqueousammonia. Negamycin which has carboxylic acid group can be adsorbed alsoby an anion exchange resin. For instance, Dowex 1X2 (trade name, DowChemical Co., U.S.A.) in OH form adsorbs negamycin and negamycinadsorbed is eluted with dilute hydrochloric acid. However, anionexchange resin having weak basic group such as Amberlite IR45 (tradename, Rohm and Haas Co., USA.) in OH form can be used for neutralizationof acidic solution of negamycin without substantial adsorption of thisantibiotic.

Mycelium in the fermented broth can be removed by filtration orcentrifugation. The fermented broth including mycelium can be alsodirectly applied to a resin column without procedure of removal of themyc'elium, if a large particle which prevents the pass through thecolumn was removed by passing through metal nets. The following is oneof method suitable for isolation of negamycin from the fermented brothof S. purpeofuscus: the fermented broth is filtered or centrifuged andthe filtrate or the supernatant is passed through a cation exchangeresin column in 70% NH, form, and after the column was washed with waternegamycin adsorbed was eluted with 2.0% NH OH in water and the eluatecontaining negamycin is concentrated under reduced pressure to removeammonia; the concentrated solution is passed through a column of Dowex1X2 in OH form and after washed with water negamycin on the resin iseluted with 0.5 N HCl; the eluate containing negamycin is neutralizedwith IR45 resin in OH form and concentrated under reduced pressure todryness, yielding a crude powder of negamycin hydrochloride; the crudepowder of the hydrochloride is dissolved in water and passed through aresin such as Amberlite CG-50 (trade name, Rohm and Haas Co., U .S.A.)in NH, form and the chromatography is developed with water or 0.1% NHOH; the fractions containing negamycin is evaporated under vacuum,yielding white powder of pure negamycin; if the purity is not enough,the chromatography is repeated. On the basis of the basic propertynegamycin can be precipitated by addition of a water-insoluble acid, forinstance, picric acid, p-hydroxyazobenzene-p'su1fonic acid etc.Negamycin crystallizes as the salt of p-hydroxyazobenzene-p'-sulfonicacid. Negamycin in these water-insoluble salts can be separated from theacids by methods known to separate a base from a water-insoluble acid,for instance, negamycin p-hydroxyazobenzene-p'-sulfonate is treated withHCl in ethanol and negamycin hydrochloride is precipitated.Water-insoluble acid such as Targitol 4, Targitol 7 (Union Carbide andCarbon Chemical Co.) and sodium laurylsulfonate can also be used forprecipitation of negamycin.

Negamycin purified by methods described above is obtained as whitepowder. It is freely soluble in water, and insoluble in methanol,ethanol, esters, benzene. Negamycin in the aqueous solution shows endabsorption, but no maximum absorption of ultraviolet light from 220 to400 m;;.. It is amphoteric, showing strong basic property and weakacidic property. Under 3500 v. and pH 1.8 buffer (formic acid-aceticacid-water in 25:75 :900 in volume) negamycin moves by 12 cm. tocathode, and taking the mobility of alanine as 1.0, the mobility ofnegamycin is 1.4. It forms acid addition salts with hydrochloric acid,

sulfuric acid, p-hydroxyazobenzene-p'-sulfonic acid, picric acid, etc.Negamycin having carboxylic acid group also forms metal salts andesters. It gives positive reactions to Ninhydrin, Rydon-Smith andnegative reactions to anthrone and Sakaguchi. When heated at 105 C. in 6N HCl for 6 hours, the hydrolysate showed more than 3 Ninhydrin products(the main products are three). Three man hydrolysis products arer-hydroxy-fl-lysine, N,N'-dimethylhydrazine and methylamine. Sarcosineand l-methylhydraginoacetic acid are obtained as minor hydrolysisproducts.

Most purified negamycin melted gradually from 75 C. and decomposed withfoaming at 110-120 C. The following result was obtained by elementalanalysis: calcd. for C H N O -2H O: C, 38.02; H, 8.51; N, 19.71; 0,33.77. Found: C, 37.78; H, 8.62; N, 18.94; 0, 31.70. As the titrationequivalent 287 was obtained. Negamycin p-hydroxyazobenzene-p-sulfonateis obtained as yelloworange crystals, M.P. ISO-182 C. withdecomposition, which shows about antibacterial activity of negamycin andwhich showed the following analytical result: calcd. for C9H2N4O4 (C H NO.;.S) 2H O: C, H,

N, 13.33; 0, 26.64; S, 7.63. Found: C, 47.09; H, 5.16; N, 13.20; 0,25.53; S, 8.01. Negamycin has three titrable pK as follows: 3.55, 8.10,8.75. In the infrared spectrum of negamycin taken with potassiumbromide, absorption bands were observed at the following wave numbers incm.- 3430, 3200, 3050, 2950, 1660, 1590, 1405, 1320, 1140, 1050, 970,890, 820, 720. Negamycin p-hydroxyazobenzene-p'-sulfonate showsabsorptions bands at the following wave numbers in cm. 3550, 3400, 3350,3250, 3200, 3050, 2950, 1730, 1690, 1600, 1560, 1510, 1440, 1430, 1400,1360, 1280-1100, 1060, 1030, 101-0, 990, 960, 945, 900, 880, 850, 800,715, 680. In this salt the band for carboxylic acid is shown at 1730 cmrThe infrared spectrum of negamycin methyl ester shows the bands at thefollowing wave numbers in cmr z 3400, 3200, 3000, 2950, 1740, 1665,1600, 1485, 1445, 1400, 1220, 1140, 1050, 1000, 960, 890, 720. The bandfor the ester group is at 1740 and 1220 cmf The structure already shownwas proposed by the present inventors for negamycin on the basis of massspectometry of negamycin methyl ester and triacetyl negamycin methylester and determination of structures of hydrolysis products.

Negamycin inhibits growth of Gram negative and positive organisms. Whenthe antibacterial effect was tested by agar streak method using meatextract-peptone agar, the following organisms were inhibited at thefollowing concentrations (one loopful amount of 20 hours nutrient brothculture was streaked; the values in parenthesis are the concentrationsof partial inhibition): Staphylococcus aureus 209p 50 ug./ml.;Staphylococcus aureus 193 12.5 ug./ml.); Staphylococcus aureus Smith 50,ag./ ml.; Sarczna lutea PCI-1001 12.5 ,LLg-/II11. (1.56 ,ag./ml.);Bacillus subtilis NRRL-B558 25 ,ag./ml. (12.5 ug./ml.); E. coli K12 3.12,ag/ml. 1.56 ug./ml.); E. coli NIH] 12.5 lg/ml. (3.12 ,ug./ml.);Shigella flexneri 12.5 ,ug./ml.; Salmonella typhosa 3.12 ,ug./ml.,Klebsiella pneumoniae PCI 602 12.5 ugjml. (6.25 ,ag./ml.), Seratiamarscescens 12.5 ug/ml. (6.25 ,ug./mL), Proteus vulgaris 0X19 6.25ag./ml.; Proteus rettgeri (GN311) 12.5 ug./ ml. (6.25 ug./ml.), Pseudomnas aerulginosa (No. 2) 12.5 ug./ml.; Pseudomonas aeruginosa No. 3. 25ug/ml. (12.5 ug./ml.); Pseudomonas fluorescens 6.25 ,ug/ml. (3.12,ag./ml.); Mycobacterium smegmatis 607 ,ugJml. When the 20 hoursnutrient broth culture was 1000 times diluted and one loopful amount wasstreaked, then the growth inhibition was observed at half concentrationsof those described above. In 0.5% peptone agar, the inhibitory elfectwas stronger than on the nutrient agar and the following organism wereinhibited at the following concentrations (values in parenthesis areconcentrations of partial inhibition): S. aureus 209p 1.56 ag./ml.; S.lutea PCI 1001 5.0 ug./ml. (2.5 ,ug./II11.; B. Sub'tilis NR'RL-B558 12.5,ag./ml.; E. coli K12 1.56 ,ag./ml.; E. coli NIH] 1.56 ,ag./ml.; S.flexneri 3.12 ag./ml.; K. pneumoniae PCI 602 6.25 ug/ml. (3.12,ag./ml.); S. typhosa 0.78 ,ag./ml.; Pr. rettgeri CN311 1.56 g./ml., S.marscescens 12.5 ,ug./rnl. (3.12 ug./ml.; Ps. aeruginosa No. 3 6.25ug/ml. Ps. aeruginosa No. 46 3.12 ug/ml. (1.56 g./ml.); M. segmatis 60750 ug/ml. It does not inhibitc. albicans at 100 ,ag/ml. The inhibitoryefiect of negamycin is not reduced by serum, for instance, E. coli wasinhibited by 3.12 ,ug/ml. of negamycin in all media containing serum at0, 5, 10, 20, 40%. Negamycin exhibits stronger antibacterial effect atalkaline side, for instance, the inhibitory concentrations against E.coli in 0.5 peptone water of various pH were as follows; pH 5.0 12.5ugjml. (3.12 g./ml.); pH 6.0 12.5 ugJml. (3.12 ag./ml.); pH 7.0 3.12ug/ml. (1.56 agJmL); pH 8.0 1.56 ,ug./ml. (0.78 g./ml.); pH 9.0 1.56gJml. (0.78 ,ug./ml.). In a cylinder plate method using 0.5 peptone agarand E. coli K12, 25 g/ml. of negamycin showed the following inhibitiondiameters at the following pH: pH 6.0 16 mm.; pH 7.0 20 mm.; pH 8.0 21mm.

Negamycin has low toxicity. The intravenous injection of 400 mg./ kg.caused death of mice. No toxic sign was observed, when 200 mg./kg. wasdaily intraperitoneally injected to mice for 30 days. It has no delayedtoxicity.

The intramuscular injection of 50 mg./kg. to rabbit gave high bloodconcentration such as 100 ig/ml. at 1 hour after the administration and80% was excreted into urine in 24 hours indicating high concentration inurine, for instance 4480 ,ug./ml. in urine taken 1-2 hours after theinjection. These results suggest the positive effect also in vivo.Really, negamycin was confirmed to be effective against infection ofPseudomonas aeruginosa No. 12, Klebsiella pneumoniae S-1802, Salmonellatyphosa 63 and Staphylococcus aureus Smith S-424 in mice. CD againstthese infections were 4.4, 5.0, 2.5 and 12.5 mg./ kg., respectively,when 10 MLD was infected intraperitoneally and negamycin wassubcutaneously injected immediately and 6 hours after the infection.

As described above, negamycin is an antibacterial substance having lowtoxicity and inhibiting Gram positive and negative bacteria includingPseudomonas. This antibiotic is easily differentiated from knownantibiotics by its analytical results, its infrared spectrum, itsamphoteric property, its behaviour on high voltage electrophoresis andits structure. Thus, negamycin is definitely a new antibiotic discoveredby the present inventors.

As shown by the structure, hydrolysis of negamycin givesl-methylhydrazino-acetic acid which was found to convert toN,N-dimethylhydrazine. 1-methyl-hydrazinoacetic acid was found by thepresent inventors to inhibit glutamic acid-pyruvate transaminase and hasliver toxicity. Negamycin has no such toxicity, however, considering thepossible hydrolysis, negamycin should be infected for short period totreat the sensitive infections. Negamycin is also used locally fortreatment of superficial infections such as infections in skin andmucous membrane. Moreover, negamycin is useful for synthesis ofnegamycin analogues.

Properties of negamycin is now clear and therefore on the basis of theproperties disclosed by the present invention processes for productionof this antibiotic is easily devised. The examples are shown below.However, this invention is not limited to these examples. This inventionincludes all modifications of processes described in this invention.

EXAMPLE 1 A medium 125 ml. containing glucose 2%, starch 2%, soybeanmeal 2%, yeast extract 0.5%, NaCl 0.25%, CaCO 0.35%, CuSOLySH O 0.0005%,MnCl -7H O 0.0005%, and ZnSO -7H O 0.0005% was placed in a shaking flaskof 500 ml. After adjusting pH to 7.0, it was sterilized at 120 C. for 20minutes. To this medium, 2.5 ml. of 2 days shaken-cultured broth of thestrain No. M890-C2 was inoculated aseptically. The medium for seedculture contained glucose 1%, starch 1%, meat extract 0.75%, peptone0.75%, NaCl 0.3%, MgSO -7H O 0.1%, CuSO -5H O 0.0007%, FeSO -7H O0.0001%, MnCl -7H O 0.0008%, and ZnSO -7H O 0.0002%. The shaking of 130strokes per minute was given during the fermentation period (5 days) andtemperature was kept at 27 C. This cultured broth was centrifuged toseparate mycelia and 6000 ml. of the filtrate was obtained from thesixty flasks. The filtrate contained 35 ngJml. of negamycin. Thefiltrate was passed through a column (diameter 30 mm.) containing 400ml. o-f Amberlite IRC-SO (70% Na form). After washed with water,negamycin was eluted with 20% ammonia in water. The active fraction of600 ml. was concentrated under reduced pressure, and 144 ml. of theconcentrated solution (pH 9.4, 800 ,ug./ ml.) was obtained.

The concentrated solution was passed through a column (diameter 16 mm.)containing 60 ml. of Dowex 1X2 (OI-I form). After washed with 200 m1. ofwater, negamycin was eluted with 0.5 N HCl. After neutralization withAmberlite IR-45 (OH form), the active fraction was dried under reducedpressure. The crude powder of negamycin hydrochloride of 300 mg. (purity21%) was obtained.

EXAMPLE 2 EXAMPLE 3 Various kinds of ion exchange resins were tested forisolation of negamycin. The broth filtrate of 200 ml. obtained by aprocedure as shown in Example 1 was passed through a column (diameter 10mm.) containing 10 ml. of a resin. The column was washed with 50 ml. ofwater, and then eluted as shown below. In all cases, no activity wasrecognized in effluents and water used for washing of the column. Theresults were as below:

Recovery of negamycin,

Ion exchange resin Type Elutiou percen;

Amberlite IRC-50 H form 2% NH40H 45 70% Na form 2% NHiOI-I 52 70% Naform 1N H014 20 70% N H; form- 2% NH4OH 55 Amberlite IR- H form 2% NH4OH45 Lewatit SP-100 do 2% NHiOH 50 EXAMPLE 4 The medium of 10 literscontaining the same constituents as shown in Example 1 was placed in aglass fermenter of 20 liters. After sterilization, 500 ml. of theshaken-cultured broth of the strain No. M890-C3 was inoculatedaseptically. The fermentation was operated at 27 C. for 90 hrs. underaeration and agitation. This cultured broth (pH 7.4) was centrifuged toseparate mycelia and 6460 ml. (41 ,lLg./IT11.) of the filtrate wasobtained. The filtrate was passed through a column (diameter 30 mm.)containing 400 ml. of Amberlite IRC-SO (70% Na form). After washed with3000 ml. of water, negamycin was eluted with 2% ammonia. The activeeluate of 400 ml. was dried under reduced pressure, and 685 mg. (purityg/ml.) of brownish powder of crude negamycin was obtained.

EXAMPLE 5 Negamycin of 102 mg. was dissolved in 8 ml. of watercontaining 15% ethylene glycol monomethyl ether. To this solution, 106mg. of sodium p-hydroxy-azobenzenep-sulfonate was added. The mixture waswarmed to 50 C. to dissolve, and then 0.5 ml. of 1 N HCl was added toadjust pH to 3. After storage in refrigerator, 49 mg. of yellow-orangecrystals was obtained. It was recrystallized from the mixed solvent(water 4.5 ml. and methanol 0.5 ml.). Twenty eight mg. of yellow-orangecrystals was obtained. M.P. -182 C. (dec.). The crystal has 34% activityof free negamycin against E. coli K12. Analysis: CalCd. forC9H20N4O4(C12H1QN2O4S)22H2OI C, 47.13, H, 5.27, N, 13.33, 0, 26.64, S,7.63; found: C, 47.09; H, 5.16, N, 13.20, 0, 25.53, S 8.01.

EXAMPLE 6 A medium (130 liters) containing glucose 3%, starch 1%,soybean meal 2%, dried yeast 0.5%, CaCO 0.35% CuSO -5H O 0.0005%, MnCl-7H O 0.0005%, and ZnSO -7H O 0.0005 was placed in a 200 litersstainless steel fermenter. After sterilization at 120 C. for 30 minutes,1.2 liters of a cultured broth of the strain No. M890C2 were inoculatedaseptically. The stirring of 200 r.p.m. and the aeration of 25 litersair per minute were given during the fermentation period (113 hrs.) andtemperature was kept at 27 C. The cultured broth of 110 liters (pH 7.4,63 ,ug./ml.) was filtered with the aid of 8 kg. of diatomaceous earthafter adjusting pH to 4.9 with 1 liter of 6 N HCl. Including the waterused for washing of the filter, totally 117 liters of the filtrate wereobtained. The filtrate was passed through a column containing 6.5 litersof Amberlite IRC-50 (70% NH, form). After washed with water, negamycinwas eluted with 0.5 -N ammonia. The active eluate of 3 liters wasconcentrated to 495 ml. (3520 ag/ml.) under reduced pressure. Theconcentrate was passed through a column containing Dowex 1X2 OH type).After washed with 350 ml. of water, negamycin was eluted with 0.5 N HCl.The active eluate of 1180 ml. was neutralized with 3 N ammonia.Yellow-brownish powder (5.5 g.) of crude negamycin (purity 294 ,ugJmg.)was obtained after drying. The overall yield was 23.4%.

EXAMPLE 7 The powder of 3.4 g. having 29.4% purity obtained in Example 6was dissolved in water of 22 ml. and adjusted to pH 8.8. This solutionwas applied to a column (diameter 26 mm.) filled with 250 ml. ofAmberlite CG50 (NH form). After washed with 500 ml. of water, 0.1% NH OHwas passed through the column. The eluate was fractionated by 20 ml.Negamycin was found in fractions 69-81. From fractions 69-73, 233 mg.(purity 90%) of pale-yellowish powder of negamycin was obtained. Fromfractions 74-77, 383 mg. of pure negamycin was obtained. From fraction78-81, 395 mg. of pure negamycin was obtained.

Included within the scope of the present invention are negamycin andacid addition salts of negamycin with organic and inorganic acids suchas hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid,nitric acid, citric acid, maleic acid, malic acid, tartaric acid,benzoic acid, cinnamic acid, ascorbic acid, acetic acid, picric acid,p-hydroxyazobenzene-p'-su1fonic acid, phytic acid,livopimaric-6,8a-cis-endosuccinic acid, sulfamic acid, glycolic acid andmandelic acid. For therapeutic purposes use is made of salts of nontoxicacids but salts of toxic acids, e.g., p-hydroxyazobenzene-p-sulfonicacid, are useful in isolation procedures, e.g. as precipitants fromaqueous solutions, and for disinfectant purposes where toxicity is notimportant.

When desired for specific purposes and rendered pharmaceuticallycompatible, there may be admixed with the compounds of present inventionother medicaments such as antihistamines, sulfa drugs (e.g.sulfadiazine, sulfabenzamide, sulfacetamide, sulfanilamide,sulfapyridine, sulfathiazole, sulfapyrazine, sulfaguanidine,sulfathalidine, sulfasuxidine, sulfisoxazole, sulfamylon,phthalylsulfacetamide, N'-3,4-dimethylbenzoylsulfanilamide,benzylsulfanilamide and N'-2-(2-quinoxalyl) sulfanilamide), lipotropicagents (particularly methionine, choline, inositol and beta-sitosteroland mixtures thereof), stimulants of the central nervous system (e.g.aspirin, salicylamide,

sodium gentisate, p-acetylaminophenol, phenacetin, codeine), laxatives(e.g. phenol-phthalein), sedatives (e.g. barbiturates, bromides), saltsof penicillin (e.g. potassium penicillin G, procaine, penicillin G,l-ephenamine pencillin G, dibenzylamine penicillin G; these combinationsare particularly useful to enable variation of the pattern of bloodlevels obtained), phenoxymethyl-penicillin and salts thereof, otherantibiotic agents (e.g. streptomycin, dihydrostreptomycin, kanamycin,bacitracin, polymixin, tyrothricin, erythromycin, chlortetracycline,oxytetracycline, tetracycline, oleandomycin, chlorarnphenicol,magnamycin, novobiocin, cycloserine, neomycin; in some cases suchcombinations attack a wider range of organisms or show synergisticeific'acy or provide decreased toxicity with equal efficacy), vitamins(e.g. vitamins A, A B B B B and members of that family, folic acid andmembers of that family, vitamins C, D D and E), hormones (e.g.cotrisone, hydrocortisone, 9-ot-fiuorocortisone,9-04-fl110l'0hYd1'0C01tiS0116, prednisone and prednisolone), anabolicagents (e.g. 11,17- dihydroxy 9 a fluoro-17-u-methyl-4-androsten-3-one;17 0c ethyl 19 nortestosterone) and antifungal agents (e.g.myconstatin).

What we claim is:

1. The process for the production of an antibiotic, designatednegamycin, which comprises cultivating a strain of Streptomycespurpeofuscus in an aqueous carbohydrate solution containing anitrogenous nutrient under aerobic conditions until substantialantibacterial activity is imparted to said solution and then recoveringsaid negamycin from said solution.

2. The process of claim 1 wherein the organism is Streptomycespurpeofuscus, A.T.C.C. 21470.

3. The process of claim 1 wherein the antibiotic negamycin is recoveredfrom an aqueous solution thereof by adsorption on a cation exchangeresin and subsequent elution.

4. The process of claim 1 wherein the antibiotic negamycin is recoveredfrom an aqueous solution thereof by adsorption on carbon and subsequentelution.

5. The process of claim 1 wherein the antibiotic negamycin is recoveredfrom an aqueous solution thereof by adsorption on strongly basic anionexchange resin and subsequent elution.

6. The process of claim 1 wherein the antibiotic negamycin is recoveredfrom an aqueous solution by precipitation with a water-insoluble acidand the subsequent separation from the acid.

7. A process according to claim 2 wherein ammonia is used for elution ofthe adsorbed antibiotic.

References Cited Hamada et al.: Journal of Antibiotics, vol. 23, No. 3,p. (1970).

A. LOUIS MONACELL, Primary Examiner R. J. WARDEN, Assistant Examiner US.Cl. X.R.

